Process for producing hydrazinomonosaccharide derivatives and use thereof

ABSTRACT

A process for producing hydrazinomonosaccharide derivatives and use of hydrazines in determining the structures of aldose and ketose monosaccharides located at the reducing ends of saccharides.

TECHNICAL FIELD

The present invention relates to a method for producing hydrazinomonosaccharide derivatives which is useful for analyses ofmonosaccharides and use thereof. Specifically, the present inventionrelates to use of hydrazines for the production of hydrazinomonosaccharide derivatives and for the determination of structures ofaldose or ketose monosaccharides at reducing ends of saccharides.

BACKGROUND ART

Saccharides (also called sugars or carbohydrates) are main components ofbiological systems. Saccharides constitute about 80% of dry weight ofplants and, either as monomers (monosaccharides) or polymers consistingof monosaccharides covalently bound each other (oligosaccharides), areindispensable components of metabolic pathways in higher animals. Inaddition, saccharides are often found as parts of larger biologicalmacromolecules (including proteins, lipids and nucleic acids).Saccharides in such various forms have numbers of important functions innature.

A means of identifying a free monosaccharide or a monosaccharide as amonomeric component of an oligosaccharide is very useful because of theimportance of saccharides in biological systems. Furthermore, a means ofidentifying a monosaccharide at a reducing end of an oligosaccharide isimportant for the structural analysis of the oligosaccharide.

Recently, a method for analyzing a structure of an oligosaccharide wasdisclosed in WO 96/17824 (JP-A 11-501901). In the method, amonosaccharide at a reducing end of an oligosaccharide is converted toan N,N′-diacetylhydrazino monosaccharide derivative, and the derivativeis identified by gas chromatography/mass spectrometry (GC/MS) or thelike. Theoretically, the types of monosaccharides at the reducing endsof all oligosaccharides can be identified according to this method.

However, there is a problem that one can use the above-mentioned methodonly for an isolated saccharide. If this method is applied to a mixtureof several kinds of saccharides, it is impossible to determine thesaccharides from which the respective resulting several kinds ofhydrazino monosaccharides derive. Thus, if a naturally occurringsaccharide is to be analyzed according to this method, it is necessaryto isolate the saccharide beforehand.

For example, since a protein or a nucleic acid itself has ultravioletabsorbance, it is possible to trace the position duringseparation/purification procedures based on the absorbance. However,since a saccharide does not have such absorbance, it is desired to labelthe saccharide in order to facilitate separation/purification. Forexample, labeling with a fluorescent dye is suitable for this purpose.If a saccharide coexists with other contaminants derived from a naturalsource (e.g., proteins, nucleic acids, etc.), the saccharide having alabel should be readily distinguished from other contaminants. Thus, inthis case, a saccharide must be selectively labeled such thatcontaminating components other than the saccharide are not labeled.

A method for labeling a reducing residue of a saccharide exemplifies amethod for selectively labeling a saccharide without labeling othercomponents derived from a natural source. Examples of reducing residuesinclude a carbonyl group at a reducing end of a saccharide and a freealdehyde group. Reactions on reducing residues of saccharides include areductive amination reaction and a hydrazidation reaction. Thesereactions are irreversible. A method in which 2-aminopyridine is used(S. Hase, T. Ibuki and T. Ikenaka, Journal of Biochemistry, 95, 197-203(1984)) exemplifies a method for labeling a saccharide using a reductiveamination reaction. A method in which Biotin-x-hydrazide (Calbiochem) isused (B. Ridley, D. Mohnen et al., Analytical Biochemistry, 249, 10-19(1997)) exemplifies a method for labeling a saccharide using ahydrazidation reaction. In the latter method, a saccharide labeled withbiotin through a hydrazide bond is prepared by forming a hydrazone by areaction of a saccharide having a reducing end with Biotin-x-hydrazideand then conducting a reduction reaction.

A free reducing end of a saccharide is utilized in the method disclosedin WO 96/17824. Therefore, it is impossible to directly use amonosaccharide isolated by labeling a reducing end of a saccharideaccording to this method for identifying the type thereof.

Furthermore, determination of the position of binding to a neighboringmonosaccharide (hereinafter also referred to as the “substitutionposition”) is desired in addition to identification of the type of amonosaccharide in order to precisely determine the structure of amonosaccharide at a reducing end of a saccharide. In other words, it isdesired to determine the position of a hydroxyl group of a neighboringmonosaccharide through which a monosaccharide at a reducing end binds.Only a technique for identifying the type of a monosaccharide at areducing end of a saccharide is disclosed in WO 96/17824. A techniquefor determining the substitution position is not mentioned therein.

A methylation analysis is known as a method for determining the positionof binding between monosaccharides constituting a saccharide. A generalmethylation analysis comprises methylation of an oligosaccharide,hydrolysis of the methylated oligosaccharide, reduction of a freemethylated monosaccharide, acetylation of a methylated alditol, and ananalysis of a partially methylated alditol acetate (PMAA) in this order.Fragmentation patterns for partially methylated alditol acetates uponmass spectrometric analyses and rules thereof have been studied indetail for a long time, and it is possible to identify the position ofan acetyl group based on the fragmentation pattern (B. Lindberg, Methodsin Enzymology, Vol. 28, pp. 178-195 (1972)). PMAAs are generated for allmonosaccharides constituting an oligosaccharide according to a generalmethylation analysis. They are usually analyzed using gaschromatography/mass spectrometry (GC/MS) equipment. The position of anacetyl group can be identified based on the fragmentation pattern upon amass spectrometric analysis. On the other hand, one has to rely onidentification by comparison with a standard substance on gaschromatography for identification of the type of the monosaccharide. Forthis purpose, it is required that all possible PMAAs for allmonosaccharides constituting an oligosaccharide have been provided asstandard substances. It requires a lot of labor to prepare possiblePMAAs for all naturally occurring monosaccharides. In addition, it ispractically impossible to conduct chromatography that can be used toseparate and identify all possible PMAAs for all naturally occurringmonosaccharides.

OBJECTS OF INVENTION

The main object of the present invention is to provide a means thatenables determination of the type and the substitution position of amonosaccharide at a reducing end of a saccharide even if the saccharideis not isolated.

SUMMARY OF INVENTION

The present inventors have studied intensively in order to achieve theabove-mentioned object. As a result, the present inventors have foundthat it is possible to determine the type of a monosaccharide at areducing end of a saccharide according to a method similar to the methodas described in WO 96/17824 by converting the saccharide into ahydrazino monosaccharide derivative even if the saccharide is notisolated. Thus, the present invention has been completed.

Accordingly, the present invention provides the following:

(1) a method for producing a hydrazino monosaccharide derivative, themethod comprising at least:

(a) reacting a saccharide having a reducing end with a hydrazine offormula (I) to produce a hydrazone:NH₂—NR¹(R²)  (I)wherein R¹ is a group other than hydrogen that has a detectable labeland/or an immobilization support as its portion or can bind to adetectable label and/or an immobilization support; the bond between R¹and N is a bond that is cleavable by a reaction that can cleave aglycosidic linkage; and R² is hydrogen or an alkyl group containing 1-8carbon atoms;

(b) reducing the hydrazone obtained in step (a) to a hydrazinoderivative; and

(c) cleaving the hydrazino derivative obtained in step (b) by thereaction that can cleave a glycosidic linkage to obtain a hydrazinomonosaccharide derivative;

(2) the method according to (1), which comprises N-acetylating thehydrazino derivative obtained in step (b) before subjecting it to step(c);

(3) the method according to (1) or (2), which comprises methylating ahydroxyl group of the hydrazino derivative obtained in step (b) or anN-acetylation product of the hydrazino derivative before subjecting itto step (c);

(4) the method according to any one of (1) to (3), wherein R¹ is an acylgroup;

(5) the method according to any one of (1) to (4), wherein R¹ is an acylgroup that has a ultraviolet or visible-absorbing substance, afluorescent dye or an immobilization support as its portion;

(6) a method for identifying a monosaccharide at a reducing end of asaccharide having a reducing end and/or for determining a position ofbinding of a monosaccharide at a reducing end to a neighboringmonosaccharide, the method comprising at least:

(a) reacting a saccharide having a reducing end with a hydrazine offormula (I) to produce a hydrazone:NH₂—NR¹(R²)  (I)wherein R¹ is a group other than hydrogen that has a detectable labeland/or an immobilization support as its portion or can bind to adetectable label and/or an immobilization support; the bond between R¹and N is a bond that is cleavable by a reaction that can cleave aglycosidic linkage; and R² is hydrogen or an alkyl group containing 1-8carbon atoms;

(b) reducing the hydrazone obtained in step (a) to a hydrazinoderivative;

(c) cleaving the hydrazino derivative obtained in step (b) by thereaction that can cleave a glycosidic linkage to obtain a hydrazinomonosaccharide derivative;

(d) completely acetylating the hydrazino monosaccharide derivativeobtained in step (c); and

(e) identifying the completely acetylated hydrazino monosaccharidederivative obtained in step (d);

(7) the method according to (6), which comprises N-acetylating thehydrazino derivative obtained in step (b) before subjecting it to step(c);

(8) the method according to (6) or (7), which comprises methylating ahydroxyl group of the hydrazino derivative obtained in step (b) or anN-acetylation product of the hydrazino derivative before subjecting itto step (c), wherein a position of binding of a monosaccharide at areducing end to a neighboring monosaccharide is determined in step (e);

(9) the method according to any one of (6) to (8), wherein theidentification in step (e) is carried out using gas chromatography/massspectrometry (GC/MS);

(10) a method for labeling a saccharide having a reducing end, themethod comprising at least:

(a) reacting a saccharide having a reducing end with a hydrazine offormula (I) to produce a hydrazone:NH₂—NR¹(R²)  (I)wherein R¹ is a group other than hydrogen that has a detectable labeland/or an immobilization support as its portion or can bind to adetectable label and/or an immobilization support; the bond between R¹and N is a bond that is cleavable by a chemical reaction that can cleavea glycosidic linkage; and R² is hydrogen or an alkyl group containing1-8 carbon atoms;

(b) reducing the hydrazone obtained in step (a) to a hydrazinoderivative; and

(c) N-acetylating the hydrazino derivative obtained in step (b);

(11) a hydrazine of formula (I) used in the method defined by any one of(1) to (10):NH₂—NR¹(R²)  (I)wherein R¹ is a group other than hydrogen that has a detectable labeland/or an immobilization support as its portion or can bind to adetectable label and/or an immobilization support; the bond between R¹and N is a bond that is cleavable by a reaction that can cleave aglycosidic linkage; and R² is hydrogen or an alkyl group containing 1-8carbon atoms;

(12) a kit for producing a hydrazino monosaccharide derivative, whichcontains the hydrazine defined by (11);

(13) a kit for identifying a monosaccharide at a reducing end of asaccharide and/or for determining a position of binding of amonosaccharide at a reducing end to a neighboring monosaccharide, whichcontains the hydrazine defined by (11);

(14) a kit for labeling a saccharide having a reducing end, whichcontains the hydrazine defined by (11);

(15) a saccharide of formula (III):R⁴—N(Ac)—NHR¹  (III)wherein Ac is an acetyl group; R¹ is a group other than hydrogen thathas a detectable label and/or an immobilization support as its portionor can bind to a detectable label and/or an immobilization support; thebond between R¹ and N is a bond that is cleavable by a chemical reactionthat can cleave a glycosidic linkage; R⁴ is a group that may have aglycosidic linkage with a saccharide and from which one hydrogen atomlinked to the C-1 position of a 1-deoxy aldose or one hydrogen atomlinked to the C-2 carbon of a 2-deoxy ketose is removed, excluding acase where R¹ is an acetyl group, and R⁴ is a group that does not have aglycosidic linkage with a saccharide and from which one hydrogen atomlinked to the C-1 position of a 1-deoxy aldose or one hydrogen atomlinked to the C-2 carbon of a 2-deoxy ketose is removed; and

(16) the saccharide according (15), wherein R¹ is an acyl group.

According to the present invention, it is possible to identify the typeof a monosaccharide at a reducing end of a saccharide even if thesaccharide is not isolated. Furthermore, a position of binding to aneighboring monosaccharide can be determined by methylating a hydroxylgroup of a hydrazino derivative as in the aspect of (7). Theidentification or determination as described above can be carried outwithout a need of separation of a reaction product from a reactionmixture by using a hydrazine that has an immobilization support as itsportion or contains a group that can bind to an immobilization support.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates thin-layer chromatography on which benzoylhydrazinederivatives of N-acetyl lactosamine which had been allowed to standunder various conditions and N-acetylation products thereof weredeveloped.

DETAILED DESCRIPTION OF THE INVENTION

A hydrazino monosaccharide derivative produced according to the methodof the present invention is used in a method for identifying amonosaccharide at a reducing end as described in WO 96/17824 (JP-A11-501901, incorporated herein by reference). According to the method asdescribed in WO 96/17824, a hydrazone is produced by reacting anisolated saccharide having a reducing end with a hydrazine, thehydrazone is reduced to a hydrazino derivative, a hydrazinomonosaccharide derivative is optionally cleaved from an oligosaccharide,the hydrazino monosaccharide derivative is acetylated to obtain anN,N′-diacetylhydrazino monosaccharide derivative, and thediacetylhydrazino monosaccharide derivative is identified by means ofGC/MS or the like.

There is no specific limitation concerning the saccharide having areducing end used in step (a) of the method for producing a hydrazinomonosaccharide derivative of the present invention. It may be anysaccharide of which the monosaccharide at the reducing end is to beidentified and/or the position of binding of the monosaccharide at thereducing end to a neighboring monosaccharide is to be determined. Suchsaccharides include monosaccharides, oligosaccharides andpolysaccharides as well as mixtures thereof. A sample containing asaccharide used in the method of the present invention may furthercontain other components derived from a natural source (proteins,nucleic acids, etc.). A method for obtaining a sample containing asaccharide used in the method of the present invention from a naturalsource is described, for example, in “Seikagaku jikken koza 4—Toshitsuno kagaku (Jo)”, edited by the Japan Biochemical Society, published onApr. 12, 1976, Tokyo Kagaku Dozin.

As used herein, the phrase “a monosaccharide at a reducing end” refersto a monosaccharide having a reducing property located at a terminus (areducing end) of a saccharide in which the C-1 position of an aldose orthe C-2 position of a ketose is not subjected to substitution. An“aldose” refers to either a free monosaccharide or a monosaccharide at areducing end of an oligosaccharide that may have an aldehyde group atthe C-1 position. A “ketose” refers to either a free monosaccharide or amonosaccharide at a reducing end of an oligosaccharide that may have aketone group at any one of internal carbon atoms along the backbone ofthe monosaccharide.

A hydrazine that is structurally different from the hydrazine used inthe method as described in WO 96/17824 is used in the method forproducing a hydrazino monosaccharide derivative of the presentinvention. Thereby, the need of isolating a saccharide to be analyzedbeforehand is eliminated. A “hydrazine” generally means a compoundgenerated by substituting an organic group for a hydrogen atom ofhydrazine (in a narrow sense, N₂H₄). A “hydrazine” used in the methodfor producing a hydrazino monosaccharide derivative of the presentinvention is any known or novel compound represented by formula (I):NH₂—NR¹(R²)  (I)wherein R¹ is a group other than hydrogen that has a detectable labeland/or an immobilization support as its portion or can bind to adetectable label and/or an immobilization support; the bond between R¹and N is a bond that is cleavable by a reaction that can cleave aglycosidic linkage; and R² is hydrogen or an alkyl group containing 1-8carbon atoms. Only one of four hydrogen atoms of hydrazine (in a narrowsense, N₂H₄) in the hydrazine used in the method as described in WO96/17824 may be replaced by an alkyl group containing 1-8 carbon atoms.

A “detectable label” is any label known in the art that facilitates thepurification of a hydrazino derivative. Examples thereof includeultraviolet or visible-absorbing labels (e.g., benzene and derivativesthereof), fluorescent labels (e.g., fluoresceine, pyrene, anthracene andderivatives thereof), and radioactive labels (e.g., radioactivehydrogen, radioactive carbon and radioactive iodine) as well as a biotinlabel and a digoxigenin label. If it is desired that a detectable labelwould be attached after a hydrazino derivative is produced, a hydrazinecontaining a group that can bind to a detectable label and a detectablegroup may exist independently. Alternatively, a group that has adetectable label as its portion may be used. An acyl group that has afluorescent dye as its portion exemplifies a group that has a detectablelabel as its portion. For example, a hydrazino derivative can be readilypurified by using a method known in the art such as normal phase highperformance liquid chromatography using fluorescence emitted from pyreneattached using 1-pyrenebutanoic acid, hydrazide (commercially availablefrom Molecular Probes) as an index. In addition, many compounds such asbiotin-hydrazide (Dojindo), benzoylhydrazine (Tokyo Kasei Kogyo) CascadeBlue hydrazide (Molecular Probes) are available as acylhydrazides havingdetectable groups.

Those skilled in the art can readily obtain a hydrazine compound to beused for labeling a detectable label by synthesizing it. For example, anacylhydrazide can be readily obtained by attaching a compound havingcarboxylate as a functional group to hydrazine (in a narrow sense, N₂H₄)using a technique used for peptide synthesis (Izumiya et al., “Peptidegousei no kiso to jikken” (1985) Maruzen). A compound having an aminogroup as a functional group can be condensed with hydrazine (in a narrowsense, N₂H₄) according to the above-mentioned method, for example, afterthe functional group is converted to carboxylate using succinicanhydride. In addition, hydrazine (in a narrow sense, N₂H₄) can beintroduced to a compound having a hydroxyl group as a functional groupby halogenation followed by a reaction with carbohydrazide(H₂N—NH—CO—NH—NH₂).

The above-mentioned techniques for synthesizing a hydrazine compound canbe applied not only in case of a detectable labeling compound but alsoin case of an immobilization support.

Any or all of reaction steps of the method of the present invention maybe conducted in a liquid phase or in a solid phase. An “immobilizationsupport” may be used for conducting various reactions on hydrazinoderivatives in a solid phase. For example, a position of binding of amonosaccharide at a reducing end to a neighboring monosaccharide can bedetermined in step (e) of the method for identifying a monosaccharide ata reducing end and/or for determining a position of binding of amonosaccharide at a reducing end to a neighboring monosaccharide of thepresent invention as described below in detail. The determination can beaccomplished by methylating a hydroxyl group of the hydrazino derivativeobtained in step (b) before subjecting the hydrazino derivative to step(c). In general, the methylation reaction involves several steps ofreaction/washing. A procedure for separating a reaction product from areaction mixture in each step can be omitted by attaching a hydrazinoderivative to an immobilization support.

If a hydrazino derivative attached to an immobilization support can bepurified, the saccharide to be used need not be isolated. Generally, ahydrazine that has an immobilization support as its portion or containsa group capable of binding to an immobilization support is used for anisolated saccharide. If a saccharide is not isolated, one can label amixture containing the saccharide with a hydrazine compound containing adetectable label, purify a hydrazino-derivatized saccharide using thelabel as an index, and attach the purified hydrazine-derivatizedsaccharide to an immobilization support. For example, if a saccharide isreacted with an excess amount of 4-aminobenzhydrazide, the saccharide ispreferentially condensed with a hydrazide group to form a hydrazone.After reduction, a saccharide hydrazino derivative can be purified usingultraviolet absorbance of a benzene ring as an index. The purifiedsaccharide hydrazino derivative can be attached through an amino groupof the derivative, for example, to an immobilization support havingN-hydroxy succinimide ester as a functional group. Immobilizationsupports include glass beads, polymer matrixes, sintered glass disks,fiber glass membranes and polymer membranes. It is usually desirablethat the immobilization support has a functional group for immobilizinga hydrazine or a saccharide hydrazino derivative. Examples of suchfunctional groups include an amino group, a carboxyl group, a hydroxylgroup and an alkyl halide group. Examples of immobilization supportshaving such functional groups include NovaSyn TG bromo Resincommercially available from Nova Biochem and Bio-Rex 70 Resincommercially available from Bio-Rad. A hydrazine having such animmobilization support as its portion is produced, for example,according to the procedure as described in Example 2. The reducing powerof the thus obtained hydrazine can be measured according to a methodknown in the art such as the Park-Johnson method (Park, J. T. andJohnson, M. J., J. Biol. Chem., 181, 149-151 (1949)).

The reaction of a saccharide having a reducing end with a hydrazine instep (a) is conducted under appropriate conditions known to thoseskilled in the art, for example, in an appropriate solvent (e.g., DMSOor acetonitrile) at 40-90° C. for 0.1-20 hours. For example, thereaction is conducted by heating in dimethyl sulfoxide (DMSO) containing10% acetic acid at 90° C. for 1 hour.

The reduction of a hydrazone to a hydrazino derivative in step (b) canbe conducted using any appropriate reducing agent known to those skilledin the art. Examples of appropriate reducing agents include boronhydride reagents, boron-centered hydrides, borane/diborane, aluminumhydride reagents, and other aluminum-centered hydrides having alkoxygroups that cause substitution with covalently bound carbon or hydrogen.Catalytic hydrogenation can be conducted using hydrogen gas and one ofvarious metals or a prepared alloy such as Raney nickel (anickel-aluminum alloy). In addition, dissolution of a reduced metal, useof an alkaline metal (lithium, sodium or potassium) and, for example,zinc, magnesium, tin, iron or mercury in a solvent (e.g., an alcohol,acetic acid, liquid ammonia or an ether such as 1,2-dimethoxyethane) aregenerally effective.

A reduction reaction is conducted in an appropriate solvent (e.g., DMSOor water) at 20 to 90° C. for 1 to 20 hours. For example, it isconducted by heating in a solution containing 2.5 M borane-dimethylaminecomplex and 30% acetic acid in DMSO at 80° C. for 1 hour or by allowingto stand in a 1 M sodium boron hydride aqueous solution at roomtemperature for 16 hours. Those skilled in the art understand that thetime required for such a reaction may be shortened or prolongeddepending on the elevation or lowering of temperature. The product andthe yield in a reduction step can be monitored using an analyticaltechnique such as proton NMR or mass spectrometry.

It is well known to those skilled in the art that steps (a) and (b) maybe carried out not only as separate steps but also as steps proceedingin parallel.

Furthermore, a hydrazino derivative may be N-acetylated after areduction reaction, if necessary. It is expected that the N-acetylationof a hydrazine derivative is effective in loss of charge and chemicalstabilization.

A hydrazino derivative is cleaved by a reaction that can cleave aglycosidic linkage to obtain a hydrazino monosaccharide derivative instep (c). The bond between R¹ and N contained in the hydrazine used instep (a) is also cleaved at the same time. Any reaction that can cleavea glycosidic linkage known to those skilled in the art can be used (see,for example, Biermann, C. J., Advances in Carbohydrate Chemistry andBiochemistry, Vol. 46, 251-271). R¹ is appropriately selected by thoseskilled in the art such that the bond between R¹ and N can be cleavedunder the selected reaction conditions. In one embodiment, R¹ is an acylgroup. In another embodiment, R¹ is an acyl group that has a detectablelabel (a fluorescent dye or an immobilization support) as its portion.The step of cleavage can be accomplished by using acidic conditions inone of various solvents such as water, an alcohol or carboxylic acid.Appropriate cleaving agents include a solution of hydrochloric acid ortrifluoroacetic acid in water, hydrochloric acid in absolute methanol,and sulfuric acid in an acetic anhydride solution. The cleavage isgenerally conducted at 50 to 110° C. for 1 to 10 hours. For example, thecleavage is conducted by heating in 5% hydrochloric acid-methanol at 90°C. for 4 hours or by heating in 4 M hydrochloric acid at 100° C. for 4hours.

A hydrazino monosaccharide derivative obtained by a method comprisingsteps (a) to (c) is represented by formula (II):R³—NH—NH—R²  (II).Therein, R³ is a 1-deoxy aldose moiety or a deoxy ketose moietycovalently bound to N in the formula. If R³ is a 1-deoxy aldose moiety,the covalent binding to N is generated through the C-1 position. If R³is a deoxy ketose moiety, the covalent binding to N is generated throughthe deoxy carbon in the sugar backbone (which carbon is originallypresent in the ketone group). In all cases, R² is hydrogen or an alkylgroup containing 1-8 carbon atoms.

Among hydrazino monosaccharide derivatives of formula (II), a hydrazinomonosaccharide derivative having a structure in which all or some ofhydroxyl groups of the 1-deoxy aldose moiety or the deoxy ketose moietyrepresented by R³ are methylated is designated as an “O-methylatedhydrazino monosaccharide derivative” in particular.

The aldose or the ketose constituting the group represented by R³ may beany monosaccharide in a free form or at a reducing end of anoligosaccharide. Examples of aldoses include the following: aldohexoseseach containing 6 carbon atoms (e.g., D-glucose, L-glucose, D-allose,D-altrose, D-galactose, D-gulose, D-idose, D-mannose and D-talose);aldopentoses each containing 5 carbon atoms (e.g., D-arabinose,D-lyxose, D-ribose and D-xylose); aldotetroses each containing 4 carbonatoms (e.g., D-erythrose and D-threose); and aldotrioses each containing3 carbon atoms (e.g., D-glyceraldehyde). Ketoses include ketohexoseseach containing 6 carbon atoms (e.g., D-fructose, D-psicose, D-sorboseand D-tagatose).

Using the hydrazino monosaccharide derivative obtained as describedabove, identification of a monosaccharide at a reducing end of asaccharide having a reducing end and/or determination of a position ofbinding of a monosaccharide at a reducing end to a neighboringmonosaccharide is carried out as follows.

A hydrazino monosaccharide derivative is completely acetylated in step(d). The term “acetylation” refers to covalent binding of one or moreacetyl groups to a molecule. A “completely acetylated” molecule is amolecule in which all of the free hydroxyl groups and the nitrogen atomsare acetylated.

Any appropriate O-acetylation reaction known in the art can be used forcompletely acetylating the derivative. Such reactions include, but arenot limited to, reactions with an acetic anhydride/pyridine mixture,acetic anhydride, zinc chloride, sodium acetate, sulfuric acid, oracetyl chloride in a pyridine solution (see, for example, Horton D. IA,“The Amino Sugars”, pp. 3-211, R. W. Jeanloz (ed.), Academic Press,1969; incorporated herein by reference).

N-acetylation takes place more readily than O-acetylation. Therefore,complete acetylation of deoxy-hydrazino alditol and deoxy-(N′-alkylhydrazino) alditol takes place under the conditions as described abovefor “O-acetylation” to generate acetyl groups on all of the nitrogenatoms and the free hydroxyl groups in the molecule.

For example, O-acetylation is carried out by adding a 2:1 mixture ofpyridine and acetic anhydride to a sample and incubating the resultingmixture at 37° C. for 16 hours.

If N-acetylation is to be conducted selectively without effectingO-acetylation, the N-acetylation is conducted, for example, by mixingwith acetyl anhydride in a weak alkaline buffer. Saturated sodiumbicarbonate is preferably used as a weak alkaline buffer. Unlessotherwise stated, as used herein, “N-acetylation” means acetylationselective for nitrogen atoms.

The hydrazino derivative obtained in step (b) may be methylated at thehydroxyl group before subjecting it to step (c) in the method of thepresent invention. As a result of the methylation, it is possible todetermine the position of binding of a monosaccharide at a reducing endto a neighboring monosaccharide (or the “substitution position”) in step(e).

The “methylation analysis” by which the position of binding betweenmonosaccharides constituting a saccharide is determined is useful fordetermining a position of binding of a monosaccharide at a reducing endto a neighboring monosaccharide. According to this method, a freehydroxyl group of a reducing sugar is first methylated completely, theresulting methylated saccharide is cleaved to generate a partiallymethylated monosaccharide in which only the hydroxyl group at theposition of binding to a neighboring monosaccharide is free, thepartially methylated monosaccharide is acetylated, and the position ofacetylation is identified. Methylation is conducted by any method knownin the art. For example, methylation can be conducted using the methodof Hakomori (S. Hakomori, J. Biochem. (Tokyo), Vol. 55, 205-208 (1964)),the DMSO-NaOH method (I. Ciucanu and F. Kerek, Carbohydrate Research,Vol. 131, 209-217 (1984)), or the method of Anumula et al. (an improvedDMSO-NaOH method) (Anumula, K. R. and Taylor, P. B., Anal. Biochem.,Vol. 203, 101-108 (1992)) (incorporated herein by reference).

According to the method for determining a position of binding of asaccharide at a reducing end to a neighboring monosaccharide of thepresent invention, if R₁ is an acyl group and the DMSO-NaOH method orthe method of Anumula et al. is used as a means of methylation, thecompletely acetylated hydrazino monosaccharide derivative cannot beidentified later in step (e) unless the nitrogen atom (N) directly boundto the sugar of the hydrazino derivative is acetylated prior tomethylation. Acetylation may be either N-acetylation or completeacetylation in this case. This is because an O-acetyl group introducedby complete acetylation is immediately detached under strongly basicconditions used for methylation and a methyl group is introduced inplace of the O-acetyl group. N-acetyl groups remain to be attached underthese conditions.

Those skilled in the art can readily carry out N-acetylation of ahydrazino derivative. For example, N-acetylation of a hydrazinooligosaccharide derivative can be conducted according to the method asdescribed in WO 96/17824 (JP-A 11-501901). Alternatively, a hydrazinooligosaccharide derivative may be completely acetylated by a method wellknown to those skilled in the art, for example, by allowing it to standin a 2:1 mixture of pyridine and acetic acid at 37° C. overnight.

The present inventors have found another unexpected effect ofacetylation of a nitrogen atom (N) directly bound to a carbon atomderived from a saccharide in a hydrazino derivative. Specifically, thechemical stability of a hydrazino derivative was increased byacetylating a nitrogen atom (N) directly bound to a carbon atom derivedfrom a saccharide in the hydrazino derivative. A phenomenon that anN-acetylated hydrazino derivative is very stable whereas a saccharidehydrazino derivative is unstable in an acidic solution was observed.Furthermore, it is additionally advantageous that an N-acetylatedhydrazino derivative results in a sharper peak or band as compared witha hydrazino derivative that is not N-acetylated. This is because anN-acetylated hydrazino derivative does not have a charge due toprotonation of a nitrogen atom and thus does not interact with a carrierupon chromatography for purification or analysis.

A monosaccharide at a reducing end of a saccharide having the reducingend can be identified by identifying, in step (e), the completelyacetylated hydrazino monosaccharide derivative obtained as describedabove. Furthermore, the position of binding of a monosaccharide at areducing end to a neighboring monosaccharide can be determined bymethylating a hydrazino derivative at a hydroxyl group as describedabove.

The derivative of the present invention may be purified prior toanalysis. Alternatively, it may not necessarily be purified if a systemin which chromatography is connected to analytical equipment such as gaschromatography/mass spectrometry (GC/MS) or liquid chromatography/massspectrometry (LC/MS) (generically called an on-line mass spectrometricanalysis) is used. The completely acetylated hydrazino monosaccharidederivative may be purified prior to or following acetylation. Thederivative is purified using preferably chromatography, more preferablyhigh performance liquid chromatography.

A completely acetylated hydrazino monosaccharide derivative can be usedfor determining the structure of the monosaccharide. A method fordetermining the structure preferably comprises separation bychromatography and a mass spectrometric analysis. More preferably,separation is accomplished by gas chromatography (GC/MS).

A derivative is detected using any appropriate technique known to thoseskilled in the art. Preferably, the detection is typically carried outusing ultraviolet absorbance at 200 nm or mass spectrometry (MS).Detection of a derivative using an on-line mass spectrometric analysissuch as GC/MS or LC/MS (i.e., directly introducing a compound separatedby chromatography into a mass spectrometer for analysis) is particularlypreferable.

In a mass spectrometric analysis, a sample in a gas state is ionized invacuo by a method such as electron impact (EI) or chemical ionization(CI), and the resulting ion is detected. In case of an on-line massspectrometric analysis, a molecule separated by chromatography isionized by electron impact (EI) or chemical ionization (CI). The amountof a sample required for analysis is usually less than 1 pmol. Forreview on basic equipment, see Cooks, R. G., Glish, G. L., McLucky, S.A. and Kaiser, R. E., Chemical and Engineering News, Mar. 25, 1991, pp.26-41 (incorporated herein by reference).

Unlike a general methylation analysis as described in the Background Artsection, it is intended to carry out a methylation analysis only for amonosaccharide at a reducing end according to the method for determininga binding position of a saccharide of the present invention. The type ofa monosaccharide at a reducing end can be identified by converting anoligosaccharide to a hydrazino derivative, cleaving the resultinghydrazino oligosaccharide derivative by a reaction that can cleave aglycosidic linkage, obtaining a hydrazino monosaccharide derivative,completely acetylating the hydrazino monosaccharide derivative andsubjecting the product to GC/MS (Bendiak, B. and Fang, T. T., Carbohydr.Res. 327, 463-481 (2000)). The position of binding of a monosaccharideat a reducing end to a neighboring monosaccharide can be determined by aprocedure almost the same as that of a general methylation analysis.Specifically, analysis is carried out by converting an oligosaccharideto a hydrazino derivative, completely methylating the resultinghydrazino oligosaccharide derivative, cleaving the completely methylatedhydrazino oligosaccharide derivative by a reaction that can cleave aglycosidic linkage, obtaining a partially methylated hydrazinomonosaccharide derivative, completely acetylating it and subjecting thepartially methylated/acetylated hydrazino monosaccharide derivative(partially methylated 1-deoxy-1-hydrazino alditol acetates, or partiallymethylated 2-deoxy-2-hydrazino alditol acetates, PMHAA) to GC/MS. Asaccharide other than the saccharide at the reducing end which may bedetected by the above-mentioned procedure as a partiallymethylated/acetylated saccharide can be distinguished from the partiallymethylated/acetylated hydrazino monosaccharide derivative based on theretention time on GC or the mass spectrum. Distinction based on a massspectrum is particularly effective. For example, a molecular ion mass ofPMHAA can be detected by determining a positive ion mass spectrum bychemical ionization using isobutane. If the type of a monosaccharide ata reducing end has been identified by a reducing end analysis, thenumber of possible molecular ion masses for the expected PMHAAs islimited to 6 at the most. Then, the peak for the PMHAA can be readilyidentified by scanning the chromatogram for the mass.

The position of an acetyl group in a PMHAA can be identified based on afragmentation pattern obtained by determining an MS/MS spectrum of adetected molecular ion.

It is basically possible to apply fragmentation patterns for PMAAs uponmass spectrometric analyses and rules thereof, which have beenconventionally examined in detail, to fragmentation patterns for PMHAAsupon MS/MS. However, there has been no report on obtainment of a PMHAA,and details of the fragmentation patterns unique to the PMHAAs upon massspectrometric analyses and rules thereof have not been examined yet atall.

A PMHAA can be produced using an N,N′-diacetylated hydrazinooligosaccharide (e.g., 1-deoxy-1-(N,N′-diacetyl hydrazino)-lactitol) asa raw material. The method disclosed in WO 96/17824 (JP-A 11-501901) canbe used as a method for producing an N,N′-diacetylated hydrazinooligosaccharide. An N,N′-diacetylated hydrazino oligosaccharide iscompletely methylated, and a partially methylated hydrazinomonosaccharide is then obtained, for example, by a glycosidiclinkage-cleaving reaction such as methanolysis. A PMHAA can be obtainedby completely acetylating the resulting partially methylated hydrazinomonosaccharide. The thus obtained PMHAA may be used as it is, or it maybe used after further purification by chromatography such as reversephase high performance liquid chromatography.

One of properties of PMHAAs is that they are less volatile. Due to thisproperty, mild distillation-removal of a solvent by nitrogen blowingwhich is required for a PMAA is not necessary. Loss of a sample is notobserved at all even if drying under reduced pressure using acentrifugation concentrator is carried out.

The present invention also relates to a hydrazine, a kit used in themethod of the present invention as described above. The kit containssaid hydrazine as an essential component, and is for producing ahydrazino monosaccharide derivative or for identifying a monosaccharideat a reducing end of a saccharide and/or for determining a position ofbinding of a monosaccharide at a reducing end to a neighboringmonosaccharide. The kit may further contain an additional reagent to beused for a reaction in each step, a reaction vessel, instructions andthe like.

EXAMPLES

The following examples further illustrate the present invention indetail but are not to be construed to limit the scope thereof.

Example 1

Preparation and Analysis of Hydrazino Glucose Derivative

(a) Preparation of Hydrazone

20 μl of a 4 μM 1-pyrenebutanoic acid, hydrazide (Molecular Probes)solution in dimethyl sulfoxide (DMSO) was added to 360 μg of glucosewhich had been dried adequately, and glucose was fully dissolved bysonication. 2 μl of acetic acid was added thereto and the mixture wasstirred adequately. The reaction mixture was then heated at 90° C. for 1hour.

(b) Reduction of Hydrazone

The reaction mixture obtained in (a) was dried using a centrifugationconcentrator. 20 μl of a solution containing 2.5 M borane-dimethylaminecomplex and 30% acetic acid in DMSO was added to the residue, and theresidue was fully dissolved by sonication. The mixture was then heatedat 80° C. for 1 hour. The reaction mixture was dried using acentrifugation concentrator. The residue was re-dissolved in 50%acetonitrile and purified using normal phase high performance liquidchromatography. The existence of pyrene-labeled glucose was confirmed bydetermining the molecular weight of the purified product using atriple-quadrupole ion-spray mass-spectrometer API-300 (Perkin-ElmerSciex) to detect a positive molecular ion (467.0).

(c) Methanolysis of Pyrene-Labeled Glucose

The purified pyrene-labeled glucose obtained in (b) (10 nmol) was placedin a glass test tube and dried. 100 μl of 5% hydrochloric acid-methanol(Nacalai Tesque) was added to the test tube. The tube was sealed andheated at 90° C. for 4 hours. The tube was opened, and the sample wasdried using a centrifugation concentrator to obtain a cleavage product.

(d) GC/MS Analysis

10 nmol of 1-deoxy-1-(N,N′-diacetyl hydrazino)-¹³C₆-D-glusitol as aninternal standard was added to the residue obtained after methanolysisof the pyrene-labeled glucose in (c), and the mixture was dried again.1-deoxy-1-(N,N′-diacetyl hydrazino)-¹³C₆-D-glusitol was preparedaccording to a known method (JP-A 11-501901; incorporated herein byreference) using ¹³C₆-D-glucose (Aldrich) as a starting material. 100 μlof a 2:1 mixture of pyridine and acetic anhydride was added to theresulting residue. After adequately stirring, the mixture was incubatedat 37° C. for 16 hours. The sample was dried using a centrifugationconcentrator. The residue was dissolved by adding 200 μl of chloroformthereto. 1 μl of the solution was analyzed by subjecting it to GC/MS bysplitless injection. GC/MS analysis was carried out as follows:

-   System: GCQ (Finnigan MAT)-   Column: DB-5 (5% diphenyl-95% dimethyl polysiloxane, 0.25 mm i.d.×30    m, 0.25 micrometer film thickness) (J & W Scientific)-   Carrier: Herium (40 cm/sec)-   Ionization: EI-   Injector temperature: 300° C.-   Column initial temperature: 90° C.-   Time program: 90° C. for 2 min, 90° C.—(24° C./min)—210° C., and    210° C.—(4° C./min)—300° C.-   Injection: 1 microliter (splitless injection)

Main peaks were observed at 16.04 minutes and 21.21 minutes in a totalion mass chromatogram. A peak was observed at 16.04 minutes in a singlemass chromatogram for a specific ion ([M-42]⁺) at m/z=448 for completelyacetylated 1-deoxy-1-(N,N′-diacetyl hydrazino)-¹²C₆-D-glusitol. Thispeak was consistent with a peak in a single mass chromatogram for aspecific ion ([M-42]⁺) at m/z=454 for completely acetylated1-deoxy-1-(N,N′-diacetyl hydrazino)-¹³C₆-D-glusitol from the internalstandard. The peak at 21.21 minutes had a main ion of m/z=302 which wasconsistent with the molecular ion of 1-pyrenebutanoic acid methyl ester.

Example 2

Hydrazine-Immobilized Support

(a) Preparation of Carbohydrazide-Immobilized NovaSyn TG Resin

360 mg of NovaSyn TG bromo Resin (Nova Biochem) was suspended in a 0.5 Mcarbohydrazide (Aldrich) solution in DMSO. The mixture was shaken atroom temperature for 16 hours. The resin was washed in sufficientamounts of DMSO, water and ethanol, treated with a 15 mg/ml cesiumacetate/dimethylformamide (DMF) solution, and then washed in sufficientamounts of DMF, water and ethanol to obtain carbohydrazide-immobilizedNovaSyn TG Resin. The reducing power of carbohydrazide being introducedwas measured according to the Park-Johnson method (Park, J. T. andJohnson, M. J., J. Biol. Chem., 181, 149-151 (1949)). As a result, areducing power corresponding to 1.1 nmol of 4-methoxyphenylhydrazinehydrochloride was observed for 1 mg of the resin.

(b) Preparation of Hydrazine-Immobilized Bio-Rex 70 Resin

200 mg of Bio-Rex 70 Resin (Bio-Rad) of which the ion type had beenconverted into a proton form was suspended in 5 ml ofN,N-dimethylformamide (DMF). 0.5 g of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)(Nacalai Tesque) was added thereto. The mixture was shaken at roomtemperature for 16 hours. The resin was washed in 20 ml of DMF. 5 ml ofa 10% hydrazine (Nacalai Tesque) solution in DMF was then added thereto.The mixture was shaken for additional 5 hours. The resin was washed insufficient amounts of DMF, water, 1 M hydrochloric acid, water andmethanol to obtain hydrazine-immobilized Bio-Rex 70 Resin. The reducingpower of hydrazine being introduced was measured according to thePark-Johnson method (Park, J. T. and Johnson, M. J., J. Biol. Chem.,181, 149-151 (1949)). As a result, a reducing power corresponding to27.6 nmol of 4-methoxyphenylhydrazine hydrochloride was observed for 1mg of the resin.

(c) Attachment of Saccharide to Resin

100 μl of a solution containing 100 mM glucose and 10% acetic acid inDMSO was added to 10 mg of the hydrazine-immobilized Bio-Rex 70 Resinobtained in (b). The mixture was heated at 90° C. for 1 hour. The resinwas washed in 50 ml of DMSO. 1 ml of a 1 M sodium boron hydride aqueoussolution was then added the resin. The mixture was allowed to stand atroom temperature for 16 hours. The resin was washed in sufficientamounts of water, 0.1 M hydrochloric acid, water, and ethanol, anddried.

(d) Acid Hydrolysis of Saccharide-Attached Resin

The dried saccharide-attached resin obtained in (c) was transferred to aglass test tube. 100 μl of 4 M hydrochloric acid was added thereto. Thetube was sealed and heated at 100° C. for 4 hours. After the tube wasopened, a supernatant was recovered and dried under reduced pressure toobtain a hydrolysis product.

(e) GC/MS Analysis

100 μl of a 2:1 mixture of pyridine and acetic anhydride was added tothe residue obtained after hydrolysis in (d). After adequately stirring,the mixture was allowed to stand at room temperature for 16 hours. Thesample was dried using a centrifugation concentrator. The residue wasdissolved by adding 200 μl of acetonitrile thereto. 1 μl of the solutionwas analyzed by subjecting it to GC/MS by splitless injection. GC/MSanalysis was carried out as follows:

-   System: GCQ (Finnigan MAT)-   Column: DB-5 (5% diphenyl-95% dimethyl polysiloxane, 0.25 mm i.d.×30    m, 0.25 micrometer film thickness) (J & W Scientific)-   Carrier: Herium (40 cm/sec)-   Ionization: EI-   Injector temperature: 300° C.-   Column initial temperature: 90° C.-   Time program: 90° C. for 2 min, 90° C.—(24° C./min)—210° C., and    210° C.—(4° C./min)—300° C.-   Injection: 1 microliter (splitless injection)

A peak was observed at 14.5 minutes in a single mass chromatogram for aspecific ion ([M-42]⁺) at m/z=448 for completely acetylated1-deoxy-1-(N,N′-diacetyl hydrazino)-D-glusitol. The mass chromatogramfor this peak was consistent with that for completely acetylated1-deoxy-1-(N,N′-diacetyl hydrazino)-D-glusitol as a standard.

Example 3

Methylation Analysis

(a) Preparation of Pyrene-Labeled Lactose

10 μl of a solution containing 400 nmol of 1-pyrenebutanoic acid,hydrazide (Molecular Probes) in DMSO was added to 50 nmol of lactosewhich had been dried adequately, and lactose was fully dissolved bysonication. 1 μl of acetic acid was added thereto and the mixture wasstirred adequately. The reaction mixture was then heated at 90° C. for 1hour. After the reaction mixture was dried using a centrifugationconcentrator, 10 μl of a solution containing 2.5 M borane-dimethylaminecomplex and 30% acetic acid in DMSO was added to the residue, and theresidue was fully dissolved by sonication. The mixture was then heatedat 80° C. for 1 hour. The reaction mixture was dried using acentrifugation concentrator. The residue was re-dissolved in 50%acetonitrile and purified using normal phase high performance liquidchromatography. The molecular weight of the purified pyrene-labeledlactose was determined using a triple-quadrupole ion-spraymass-spectrometer API-300. As a result, a positive molecular ion (629.3)was observed.

(b) Methanolysis of Pyrene-Labeled Lactose

The purified pyrene-labeled lactose obtained in (a) (20 nmol) was placedin a glass test tube and dried. 100 μl of 5% hydrochloric acid-methanol(Nacalai Tesque) was added to the test tube. The tube was sealed andheated at 90° C. for 4 hours. The tube was opened, and the sample wasdried using a centrifugation concentrator to obtain a cleavage product.

(c) GC/MS Analysis

100 μl of a 2:1 mixture of pyridine and acetic anhydride was added tothe residue obtained after methanolysis of the pyrene-labeled lactose.After adequately stirring, the mixture was incubated at 37° C. for 16hours. The sample was dried using a centrifugation concentrator. Theresidue was dissolved by adding 200 μl of acetonitrile thereto. 1 μl ofthe solution was analyzed by subjecting it to GC/MS by splitlessinjection. GC/MS analysis was carried out as follows:

-   System: GCQ (Finnigan MAT)-   Column: DB-5 (5% diphenyl-95% dimethyl polysiloxane, 0.25 mm i.d.×30    m, 0.25 micrometer film thickness) (J & W Scientific)-   Carrier: Herium (40 cm/sec)-   Ionization: CI (Isobutane)-   Injector temperature: 300° C.-   Column initial temperature: 90° C.-   Time program: 90° C. for 2 min, 90° C.—(24° C./min)—210° C., and    210° C.—(4° C./min)—300° C.-   Injection: 1 microliter (splitless injection)

Main peaks were observed at 8.1 minutes, 14.6 minutes and 20.1 minutesin a total ion mass chromatogram. A peak was observed at 14.6 minutes ina single mass chromatogram for a positive molecular ion ([M+H]⁺) atm/z=491 for completely acetylated 1-deoxy-1-(N,N′-diacetylhydrazino)-D-glusitol. Both the retention time and the mass spectrumwere consistent with those for the completely acetylated1-deoxy-1-(N,N′-diacetyl hydrazino)-D-glusitol as a standard.

(d) Methylation of Pyrene-Labeled Lactose

100 nmol of the purified pyrene-labeled lactose obtained in (a) wasplaced in a screw-capped glass test tube and dried under reducedpressure. 200 μl of a 2:1 mixture of pyridine and acetic anhydride wasadded to the residue. After adequately stirring, the mixture was allowedto stand at room temperature for 16 hours. The sample was dried using acentrifugation concentrator. 100 μl of methanol was added to theresidue, and the mixture was dried again. The thus obtained sample wassubjected to complete methylation treatment of the pyrene-labeledlactose using the method of Anumula et al. (Anumula, K. R. and Taylor,P. B., Anal. Biochem., 203, 101-108 (1992)). A product obtained afterextraction with chloroform was dried under reduced pressure.

(e) Methanolysis of Methylated Pyrene-Labeled Lactose

The methylated pyrene-labeled lactose obtained in (d) (corresponding to50 nmol) was placed in a glass test tube and dried. 100 μl of 5%hydrochloric acid-methanol (Nacalai Tesque) was added to the test tube.The tube was sealed and heated at 90° C. for 4 hours. The tube wasopened, and the sample was dried using a centrifugation concentrator toobtain a cleavage product.

(f) GC/MS Analysis

100 μl of a 2:1 mixture of pyridine and acetic anhydride was added tothe residue obtained in (e) after methanolysis of the methylatedpyrene-labeled lactose. After adequately stirring, the mixture wasallowed to stand at room temperature for 16 hours. The sample was driedusing a centrifugation concentrator. The residue was dissolved by adding200 μl of acetonitrile thereto. 1 μl of the solution was analyzed bysubjecting it to GC/MS by splitless injection. GC/MS analysis wascarried out as follows:

-   System: GCQ (Finnigan MAT)-   Column: DB-5 (5% diphenyl-95% dimethyl polysiloxane, 0.25 mm i.d.×30    m, 0.25 micrometer film thickness) (J & W Scientific)-   Carrier: Herium (40 cm/sec)-   Ionization: CI(Isobutane)-   Injector temperature: 300° C.-   Column initial temperature: 90° C.-   Time program: 90° C. for 2 min, 90° C.—(24° C./min)—210° C., and    210° C.—(4° C./min)—300° C.-   Injection: 1 microliter (splitless injection)

An m/z value of 393 corresponding to a positive molecular ion peak([M+H]⁺) for4-O-acetyl-1-deoxy-2,3,5,6-O-tetramethyl-1-(N,N′-diacetyl-N′-methylhydrazino)-D-glusitolwas observed when the main peak observed at 11.3 minutes was subjectedto mass spectrometry.

Example 4

Preparation of Partially Methylated/Acetylated Hydrazino MonosaccharideDerivative (Partially Methylated 1-deoxy-1-hydrazino Alditol Acetates,PMHAA)

10 μmol each of lactose(Galβ1-4Glc) (Nacalai Tesque), mannobiose(Manα1-2Man) (Dextra Laboratories), mannotriose (Manα1-6[Manα1-3]Man)(Dextra Laboratories) and N-acetyl lactosamine (Galβ1-4GlcNAc)(Seikagaku Corporation) was converted to an N,N′-diacetylated hydrazinoderivative according to the method as described in WO 96/17824 (JP-A11-501901). 1 μmol each of N,N′-diacetylated hydrazino derivative of therespective oligosaccharides was completely methylated according to themethod of Anumula et al. (supra). The resulting completely methylatedN,N′-diacetylated hydrazino oligosaccharide derivative was placed in aglass test tube and dried. 500 μl of 5% hydrochloric acid-methanol(Nacalai Tesque) was added to the test tube. The tube was sealed andheated at 90° C. for 4 hours. The tube was opened, and the sample wasdried using a centrifugation concentrator to obtain a cleavage product.300 μl of a 2:1 mixture of pyridine and acetic anhydride was added tothe residue obtained after methanolysis. After adequately stirring, themixture was incubated at 37° C. for 2 hours. The sample was dried usinga centrifugation concentrator. The residue was dissolved by adding 1000μl of an acetonitrile aqueous solution thereto and each PMHAA wasrecovered.

GC/MS Analysis

1 μl of each PMHAA solution in acetonitrile (corresponding to 1 nmol ofthe starting material) was analyzed by subjecting it to GC/MS bysplitless injection. GC/MS analysis was carried out as follows:

-   System: GCQ (Finnigan MAT)-   Column: DB-5 (5% diphenyl-95% dimethyl polysiloxane, 0.25 mm i.d.×30    m, 0.25 micrometer film thickness) (J & W Scientific)-   Carrier: Herium (40 cm/sec)-   Ionization: CI (Isobutane)-   Injector temperature: 300° C.-   Column initial temperature: 90° C.-   Time program: 90° C. for 2 min, 90° C.—(24° C./min)—210° C., and    210° C.—(4° C./min)—300° C.-   Injection: 1 microliter (splitless injection)

Plural peaks were observed for each PMHAA sample upon GC/MS analysis.The peaks for PMHAAs were identified based on positive molecular ionsfound in the respective chemical ionization mass spectra.

The results confirmed the following:4-O-acetyl-1-deoxy-2,3,5,6-O-tetramethyl-1-(N,N′-diacetyl-N′-methylhydrazino)-D-glucitolresulted from lactose;2-O-acetyl-1-deoxy-3,4,5,6-O-tetramethyl-1-(N,N′-diacetyl-N′-methylhydrazino)-D-mannitolresulted from mannobiose;3,6-O-diacetyl-1-deoxy-2,4,5-O-trimethyl-1-(N,N′-diacetyl-N′-methylhydrazino)-D-mannitolresulted from mannotriose; and4-O-acetyl-1,2-dideoxy-3,5,6-O-trimethyl-2-(N-methylacetoamido)-1-(N,N′-diacetyl-N′-methylhydrazino)-D-glucitolresulted from N-acetyl lactosamine.

Upon GC/MS analysis of4-O-acetyl-1-deoxy-2,3,5,6-O-tetramethyl-1-(N,N′-diacetyl-N′-methylhydrazino)-D-glucitolobtained from lactose, a positive molecular ion peak ([M+H]⁺) at m/z=393was detected in the chemical ionization mass spectrum for the peak at11.4 minutes. Upon GC/MS analysis of2-O-acetyl-1-deoxy-3,4,5,6-O-tetramethyl-1-(N,N′-diacetyl-N′-methylhydrazino)-D-mannitolobtained from mannobiose, a positive molecular ion peak ([M+H]⁺) atm/z=393 was detected in the chemical ionization mass spectrum for thepeak at 11.1 minutes. Upon GC/MS analysis of3,6-O-diacetyl-1-deoxy-2,4,5-O-trimethyl-1-(N,N′-diacetyl-N′-methylhydrazino)-D-mannitolobtained from mannotriose, a positive molecular ion peak ([M+H]⁺) atm/z=421 was detected in the chemical ionization mass spectrum for thepeak at 13.1 minutes. Upon GC/MS analysis of4-O-acetyl-1,2-dideoxy-3,5,6-O-trimethyl-2-(N-methylacetoamido)-1-(N,N′-diacetyl-N′-methylhydrazino)-D-glucitolobtained from N-acetyl lactosamine, a positive molecular ion peak([M+H]⁺) at m/z=434 was detected in the chemical ionization massspectrum for the peak at 14.5 minutes.

Example 5

Preparation and Methylation Analysis of Ultraviolet Absorbance-LabeledHydrazide Derivative

25 μl of a solution containing 25 μmol of benzoylhydrazine (Tokyo KaseiKogyo) in dimethyl sulfoxide (DMSO) was added to 5 μmol of glucose(Nacalai Tesque), sophorose (Glcβ1-2Glc, Sigma), laminaribiose(Glcβ1-3Glc, Seikagaku Corporation), maltose (Glcα1-4Glc, Kanto Kagaku)or isomaltose (Glcα1-6Glc, Seikagaku Corporation) which had been driedadequately, and the saccharide was fully dissolved by sonication. 2.5 μlof acetic acid was added thereto and the mixture was stirred adequately.The reaction mixture was then heated at 90° C. for 1 hour. The reactionmixture was dried using a centrifugation concentrator. 50 μl of asolution containing 2.5 M borane-dimethylamine complex and 30% aceticacid in DMSO was added to the residue, and the residue was fullydissolved by sonication. The mixture was then incubated at 37° C. for 16hours. The reaction mixture was dried using a centrifugationconcentrator. Addition of acetonitrile and drying were repeated. Theresidue was dissolved in 100 μl of water. Excess reagents were removedby three rounds of extraction with 300 μl of water-saturated ethylacetate. The method as described in WO 96/17824 (JP-A 11-501901) wasused for carrying out N-acetylation. After desalting using Dowex 50W-X8(Muromachi Kagaku) followed by further purification using HPLC,N-acetylated benzoylhydrazine saccharide derivatives were obtained. HPLCwas carried out as follows:

-   Pump: LC6A (Shimadzu)-   Column: Asahipak NH2P-50 (4.6 mm i.d.×250 mm) (Showa Denko)-   Solvent A: Acetonitrile/water, 95:5-   Solvent B: Acetonitrile/water, 1:1-   Flow rate: 1 ml/min-   Temperature: 40° C.-   Gradient: 0-100% Solvent B in 30 min-   Detection: Absorbance at 270 nm

1 μmol each of the N-acetylated benzoylhydrazine saccharide derivativeswas completely methylated according to the method of Anumula et al.(supra). A {fraction (1/20)} amount of the resulting completelymethylated N-acetylated benzoylhydrazine saccharide derivative wasplaced in a glass test tube and dried. 100 μl of a 80% acetic acidaqueous solution containing 0.5 M hydrochloric acid was added to thetest tube. The tube was sealed and heated at 100° C. for 6 hours. Thetube was opened, and the sample was dried using a centrifugationconcentrator to obtain a cleavage product. 200 μl of a 2:1 mixture ofpyridine and acetic anhydride was added to the residue. After adequatelystirring, the mixture was incubated at 37° C. for 16 hours. The samplewas dried using a centrifugation concentrator. The residue was dissolvedby adding 100 μl of an acetonitrile aqueous solution thereto to recoverPMHAA.

GC/MS Analysis

1 μl of the PMHAA solution in acetonitrile (corresponding to 1 nmol ofthe starting material) was analyzed by subjecting it to GC/MS bysplitless injection. GC/MS analysis was carried out as follows:

-   System: GCQ (Finnigan MAT)-   Column: DB-5 (5% diphenyl-95% dimethyl polysiloxane, 0.25 mm i.d.×30    m, 0.25 micrometer film thickness) (J & W Scientific)-   Carrier: Herium (40 cm/sec)-   Ionization: CI (Isobutane)-   Injector temperature: 300° C.-   Column initial temperature: 90° C.-   Time program: 90° C. for 2 min, 90° C.—(24° C./min)—210° C., and    210° C.—(12° C./min)—300° C.-   Injection: 1 microliter (splitless injection)

Plural peaks were observed in a total mass chromatogram for each PMHAA.A single peak was observed at 9.2 minutes in a single mass chromatogramfor a positive molecular ion ([M+H]⁺) at m/z=393 for the expected PMHAAfor the sample from N-acetylated benzoylhydrazine glucose. Single peakswere observed at 9.4 minutes, 9.5 minutes, 9.6 minutes and 10.2 minutesin single mass chromatograms for positive molecular ions ([M+H]⁺) atm/z=393 for the expected PMHAAs for the remaining four samples fromN-acetylated benzoylhydrazine sophorose, N-acetylated benzoylhydrazinelaminaribiose, N-acetylated benzoylhydrazine maltose and N-acetylatedbenzoylhydrazine isomaltose, respectively. The mass spectra for PMHAApeaks for respective samples were clearly different each other among thesamples. Accordingly, it was demonstrated that the position of an acetylgroup can be identified based on the difference in a mass spectrum or afragmentation pattern of PMHAA.

Example 6

Preparation and Stability Test of N-acetylated Labeled HydrazinoDerivative

100 μl of a solution containing 100 μmol of benzoylhydrazine (TokyoKasei Kogyo) in DMSO was added to 50 μmol of N-acetyl lactosamine(Seikagaku Corporation, hereinafter referred to as LN) which had beendried adequately, and the saccharide was fully dissolved by sonication.80 μl of DMSO and 20 μl of acetic acid were added thereto and themixture was stirred adequately. The reaction mixture was then heated at90° C. for 1 hour. The reaction mixture was dried using a centrifugationconcentrator while heating. 500 μl of a 25% acetonitrile aqueoussolution containing 2 M sodium boron hydride (Nacalai Tesque) was addedto the residue, and the mixture was adequately stirred and allowed tostand at room temperature overnight. 500 μl of pure water was added tothe reaction mixture. The reaction mixture was then neutralized byadding acetic acid dropwise to make the pH of the solution to about 5.800 μl of the neutralized solution was placed in a test tube. 1 ml ofpure water was further added thereto for dilution. The method asdescribed in WO 96/17824 (JP-A 11-501901) was used for carrying outN-acetylation. After desalting using Dowex 50W-X8 (Muromachi Kagaku)followed by further purification using HPLC, an N-acetylation product ofa benzoylhydrazine derivative of LN (hereinafter referred to as LNbenzoylhydrazine derivative) was obtained. On the other hand, an LNbenzoylhydrazine derivative was prepared without N-acetylation. 100 μlof a solution containing 100 μmol of benzoylhydrazine (Tokyo KaseiKogyo) in DMSO was added to 50 μmol of LN (Seikagaku Corporation) whichhad been dried adequately, and the saccharide was fully dissolved bysonication. 80 μl of DMSO and 20 μl of acetic acid were added theretoand the mixture was stirred adequately. The reaction mixture was thenheated at 90° C. for 1 hour. The reaction mixture was dried using acentrifugation concentrator while heating. 500 μl of a 25% acetonitrileaqueous solution containing 2 M sodium boron hydride was added to theresidue, and the mixture was adequately stirred and allowed to stand atroom temperature overnight. 500 μl of pure water was added to thereaction mixture. The reaction mixture was then neutralized by addingacetic acid dropwise to make the pH of the solution to about 5. 200 μlof the neutralized reaction mixture was dried using a centrifugationconcentrator and purified using HPLC to obtain an LN benzoylhydrazinederivative. HPLC was carried out as follows:

-   Pump: LC6A (Shimadzu)-   Column: Asahipak NH2P-50 (4.6 mm i.d.×250 mm) (Showa Denko)-   Solvent A: Acetonitrile/water, 95:5-   Solvent B: Acetonitrile/water, 1:1-   Flow rate: 1 ml/min-   Temperature: 40° C.-   Gradient: 0-100% Solvent B in 30 min-   Detection: Absorbance at 270 nm

25 nmol each of the purified LN benzoylhydrazine derivative and theN-acetylation product thereof was dissolved in 5 μl of a 10, 30 or 50%acetic acid solution in DMSO. The solution was incubated at 37° C.overnight. The solution was dried and re-dissolved in 5 μl of purewater. 0.5 μl of the solution was spotted onto HPTLC (Merck) anddeveloped using a 80% acetonitrile aqueous solution. Neutral sugars weredetected according to the orcinol-sulfuric acid method.

Results of thin-layer chromatography are shown in FIG. 1. The followingsamples were developed in the lanes in FIG. 1: lane 1: the untreated LNbenzoylhydrazine derivative; lane 2: the LN benzoylhydrazine derivativeincubated in a solution containing 10% acetic acid in DMSO; lane 3: theLN benzoylhydrazine derivative incubated in a solution containing 30%acetic acid in DMSO; lane 4: the LN benzoylhydrazine derivativeincubated in a solution containing 50% acetic acid in DMSO; lane 5: theuntreated N-acetylated LN benzoylhydrazine derivative; lane 6: theN-acetylated LN benzoylhydrazine derivative incubated in a solutioncontaining 10% acetic acid in DMSO; lane 7: the N-acetylated LNbenzoylhydrazine derivative incubated in a solution containing 30%acetic acid in DMSO; and lane 8: the N-acetylated LN benzoylhydrazinederivative incubated in a solution containing 50% acetic acid in DMSO.

The LN benzoylhydrazine derivative was converted under acidic conditionsinto a substance that exhibited lower mobility on TLC, whereas no changewas observed for the N-acetylated LN benzoylhydrazine derivative. Thus,the chemical stability of the N-acetylated benzoylhydrazine derivativewas demonstrated.

INDUSTRIAL APPLICABILITY

The present invention provides a means that enables determination of thetype and the substitution position of a monosaccharide at a reducing endof a saccharide even if it is not isolated.

1. A method for determining a position of binding of a monosaccharide ata reducing end to a neighboring monosaccharide, the method comprising atleast: (a) reacting a saccharide having a reducing end with a hydrazineof formula (I) to produce a hydrazone:NH₂—NR¹(R²)  (I) wherein R¹ is a group other than hydrogen that has adetectable label and/or an immobilization support as its portion or canbind to a detectable label and/or an immobilization support; the bondbetween R¹ and N is a bond that is cleavable by a reaction that cancleave a glycosidic linkage; and R² is hydrogen or an alkyl groupcontaining 1-8 carbon atoms; (b) reducing the hydrazone obtained in step(a) to a hydrazino derivative; (c) N-acetylating the hydrazinoderivative obtained in step (b); (d) methylating a hydroxy group of thehydrazino derivative obtained in step (c); (e) cleaving the hydrazinoderivative obtained in step (d) by the reaction that can cleave aglycosidic linkage to obtain a hydrazino monosaccharide derivative; (f)completely acetylating the hydrazino monosaccharide derivative obtainedin step (e); and (g) identifying the acetylated hydroxyl group in thecompletely acetylated hydrazino monosaccharide derivative obtained instep (d), and determining a position of binding of a monosaccharide at areducing end to a neighboring monosaccharide.
 2. The method according toclaim 1, wherein the identification in step (e) is carried out using gaschromatography/mass spectrometry CGC/MS).